PROJECT SUMMARY Organ transplantation is a life-saving procedure for patients with end-organ damage. Nearly 300,000 individuals die each year awaiting transplant. Despite tremendous advances, long-term graft survival remains disappointing because current immunosuppressive regimens are unable to prevent chronic rejection and require life-long treatment, placing patients at risk for infections, cancer, and heart disease. B cells are central to the development of antibody-mediated rejection and chronic rejection. However, B cells have also been shown to play a protective role. Five large independent studies unexpectedly revealed a ?B cell signature? in tolerant transplant recipients, uncovering a key regulatory role for B cells. Yet very little is known about how regulatory B cells (Breg) suppress the alloimmune response, or how they differ from pathogenic B cells. Currently there are no phenotypic, transcription factor, or lineage markers that are unique to Breg. Furthermore, Breg appear to inhibit inflammation and disease through multiple distinct pathways involving cytokines and cell surface markers. Thus, our current challenge is to comprehend the diversity of B cell subsets that possess regulatory capacity and the key decision points that determine their fate as regulatory versus pro-inflammatory. TIM-1 is an important physiological receptor of Breg, providing a crucial checkpoint to ensure peripheral tolerance is maintained. Recognition of apoptotic cells by TIM-1 promotes B cell IL-10, and a loss of function mutation of TIM-1 leads to a defect in Bregs, and heightened auto- and allo-immunity. TIM-1+, but not TIM-1-, B cells can transfer allograft tolerance, and their suppressive capacity is dependent upon TIM-1 signaling. We have now found that in addition to IL-10, TIM-1 signaling promotes the expression of many molecules with known coinhibitory activity. Our data further reveals AhR to be a key putative transcriptional regulator of Breg, downstream of TIM-1. The objective of this proposal is to map the regulatory network that controls Breg development and function, focusing on TIM-1 and AhR as key regulators. Our central hypothesis is that Bregs are a functionally and phenotypically diverse set of cells that are controlled by a common core network. Dr. Melissa Yeung is a transplant nephrologist at the Brigham & Women's Hospital/Harvard Medical School (BWH/HMS) with a strong commitment to the field of transplant immunology. Her long- term career goal is to become an independent investigator with a specific focus on understanding the molecular mechanisms governing B cell-mediated transplant tolerance. The support of the K08 award will allow Dr. Yeung to achieve the following training objectives: 1) investigate the mechanisms by which the TIM-1/AhR axis mediates Breg in alloimmunity; 2) acquire proficiency in transcriptomics research; and 3) become an expert in B cell immunoregulatory pathways. In order to accomplish these objectives and investigate our hypothesis, Dr. Yeung will first examine the role of TIM-1/AhR in regulating the function of Breg upon physiological induction and through ligation of a ?tolerogenic? anti-TIM-1 antibody (Aim1). She will do this using a network-based, transcriptomic approach to construct a model of key immunoregulatory circuits controlled by TIM- 1/AhR. To validate her model, she will then examine the functional roles of nodal regulators. Lastly, she will investigate the in vivo mechanisms by which Bregs suppress the alloimmune response (Aim2). Completion of the proposed research will provide fundamental insights into the signals involved in the ontogeny, reactivity, diversification and function of Breg, and how these pathways differentiate them from pathogenic B cells. Potential clinical applications that could result from this work include the use of Breg for cell therapy, the design of therapeutics to target pathogenic B cells whilst sparing Bregs, and the use of a Breg signature to guide immunosuppressive therapy. Dr. Yeung will be closely guided by her primary mentor Dr. Vijay Kuchroo, Professor of Neurology at BWH, Director of the Evergrande Center for Immunologic Disease at HMS and Associate Member of the Broad Institute, who has vast experience in transcriptomic and immunology research. To complement his expertise, we have formed a multi-disciplinary team of scientific advisors and collaborators: 1) Dr. David Rothstein, Professor at the Thomas E. Starzl Transplantation Institute, University of Pittsburgh, who is an expert in regulatory B cells and transplant immunology; and 2) Dr. Joseph Bonventre, Professor and Chief of the BWH Renal Division, who has extensive knowledge in KIM-1/TIM-1 biology. Dr Yeung will also draw upon the wealth of the BWH/HMS research environment, including the Evergrande Center for Immunologic Disease, the Broad Institute, the Renal Division, and the Transplantation Research Center. Dr. Yeung's proposed K08 study, mentorship team, career development plan, and collaborative research environment will catalyze her scientific productivity and provide her with a strong foundation as a future independent investigator and leader in transplantation.